Monoclonal antibodies (MAbs) produced from hybridoma cell lines were first described by Kohler and Milstein (Kohler, G. and Milstein, C. [1975] Nature (London) 256:495-497). Due to their high specificity for a single antigen, MAbs have been widely used as research tools, as components of medical diagnostic tests, and as the basis for immunoaffinity purification procedures. They also show promise as agents for in vivo human therapeutic or diagnostic use.
At the current state of the art, most hybridomas are derived from mouse B lymphocytes, and, consequently, the resulting MAbs are murine imunoglobulins. These immunoglobulins are typically of the IgG class, with subclass IgGl being the most common. The hybridomas are grown in the peritoneal cavity of mice or in mass culture, and the MAbs are isolated from the resulting ascites fluid or tissue culture fluid, respectively.
For most applications, and especially for human in vivo use, MAbs must be purified. This can be accomplished by a number of well-known methods, including ion exchange chromatography, adsorption chromatography, and affinity chromatography. Of these methods, affinity chromatography on a matrix consisting of Staphylococcal protein A immobilized to agarose beads, and sold, for example, as Protein A-SEPHAROSE.RTM. (Pharmacia AB, Uppsala, Sweden), has been found to give the most highly purified MAb (Manil, L., Motte, P., Pernas, P., Troalen, F., Bohuon, C. and Bellet, D. [1986] Journal of Immunological Methods 90:25-37). This procedure exploits the fact that murine IgG binds to Protein A-SEPHAROSE.RTM. at pH&gt;8.0, but does not bind at pH&lt;3.0. Typically, the pH of the MAb-containing solution is adjusted to 8.5 and passed over a column of Protein A-SEPHAROSE.RTM.. With the MAb bound to the proteins A, contaminants are washed from the column with a pH 8.5 buffer. Finally, the purified MAb is eluted by passing a pH 3.0 buffer over the column. The purity of the eluted antibody generally exceeds 90%.
The usefulness of Protein A-SEPHAROSE.RTM. for the purification of murine-derived MAbs of subclass IgGl, however, is extremely limited. This is due to the exceptionally low affinity of the IgGl subclass for this material, which results in a low binding capacity (defined as mg IgG/ml gel) for MAbs of subclass IgGl. Seppala et al. (Seppala, I., Sarvas, H., Peterfy, F. and Makela, O. [1981] Scand. J. Immunol. 14:335-342) reported that IgGl from mouse serum eluted from Protein A-SEPHAROSE.RTM. at pH 6.0-7.0, higher than the pH 3.5-5.0 at which the other IgG subclasses elute. Similarly, MacKenzie et al. (MacKenzie, M. R., Warner, and Mitchell, G. F. [1978] J. Immunol. 120:1493-1496) found that murine IgGl, again from polyclonal serum, eluted from Protein A-SEPHAROSE.RTM. columns at a lower concentration of sodium thiocyanate (0.5 M) than was required to elute the other subclasses (1.5-2.0 M). Consistent with the properties of polyclonal serum, Protein A-SEPHAROSE.RTM. has a lower binding capacity for MAbs of murine subclass IgGl than for MAbs of the other IgG subclasses, although the affinities of individual IgGl MAbs tend to vary somewhat (Manil et al. supra, Stephensen, J. R., Lee, J. M. and Wilton-Smith, P. D. [1984] Anal. Biochem. 142:189-195). In many cases, standard affinity chromatography procedures give a very low recovery of IgGl MAbs, and purification by more difficult and less effective methods is sometimes required.
Other commercially available matrices consisting of protein A immobilized to agarose, such as AFFI-GEL.RTM. Protein A (Bio-Rad Laboratories, Richmond, Calif.) and protein A-agarose (Pierce Chemical Company Rockford, Ill.) appear to exhibit, like Protein A-SEPHAROSE.RTM., low binding capacity for murine MAbs of subclass IgGl when the purification conditions of Ey et al. (Ey, P. L., Prowse, S. J. and Jenkin, C. R. [1978] Immunochemistry 15:429-436) are used. These limitations have been recognized by commercial suppliers who have attempted to overcome them by the use of proprietary binding solutions such as the MAPS II system (Bio-Rad Laboratories) and the MOONPURE.TM. system (Pierce Chemical Company, Rockford, Ill.). However, these solutions are expensive, and their components may not be compatible with MAbs intended for human in vivo use. Also, some samples, such as tissue culture fluid, cannot be chromatographed directly, but require instead involved preparation before addition to the column.